CD95-mediated cell signaling in cancer: mutations and post-translational modulations

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Cellular and Molecular Life Sciences ISSN 1420-682XVolume 69Number 8 Cell. Mol. Life Sci. (2012) 69:1261-1277DOI 10.1007/s00018-011-0866-4

CD95-mediated cell signaling in cancer:mutations and post-translationalmodulations

Sébastien Tauzin, Laure Debure, Jean-François Moreau & Patrick Legembre

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REVIEW

CD95-mediated cell signaling in cancer: mutationsand post-translational modulations

Sebastien Tauzin • Laure Debure •

Jean-Francois Moreau • Patrick Legembre

Received: 5 September 2011 / Revised: 10 October 2011 / Accepted: 14 October 2011 / Published online: 1 November 2011

� Springer Basel AG 2011

Abstract Apoptosis has emerged as a fundamental pro-

cess important in tissue homeostasis, immune response,

and during development. CD95 (also known as Fas), a

member of the tumor necrosis factor receptor (TNF-R)

superfamily, has been initially cloned as a death receptor.

Its cognate ligand, CD95L, is mainly found at the plasma

membrane of activated T-lymphocytes and natural killer

cells where it contributes to the elimination of transformed

and infected cells. According to its implication in the

immune homeostasis and immune surveillance, and since

several malignant cells of various histological origins

exhibit loss-of-function mutations, which cause resistance

towards the CD95-mediated apoptotic signal, CD95 has

been classified as a tumor suppressor gene. Nevertheless,

this assumption has been recently challenged, as in certain

pathophysiological contexts, CD95 engagement transmits

non-apoptotic signals that promote inflammation, carcino-

genesis or liver/peripheral nerve regeneration. The focus of

this review is to discuss these apparent contradictions of

the known function(s) of CD95.

Keywords Fas � Oncogene � Tumor suppressor �

Lipid rafts � ALPS

Abbreviations

ASM Acid sphingomyelinase

ADAM A disintegrin and metalloproteinase domain

ALPS Autoimmune lymphoproliferative syndrome

DD Death domain

FADD Fas-associating protein with a death domain

GC Germinal center

LOH Loss of heterozygosity

MAPK Mitogen-activated protein kinase

MMP Matrix metalloproteinase

NF-jB Nuclear factor Kappa B

TNF Tumor necrosis factor

Implementation of a CD95-mediated signaling pathway

CD95: the so-called death receptor

APT-1 (also known as CD95, Fas, or APO-1) gene spans

approximately 25 kb on human chromosome 10 [1]. The

gene consists of nine exons in which exon 6 encodes the

transmembrane domain (Fig. 1a). CD95 is a type I trans-

membrane protein that belongs to the tumor necrosis factor

(TNF) receptor family characterized by cysteine-rich

extracellular domains. CD95 is resolved under denaturing

conditions between 40 and 50 kDa in SDS-PAGE. This

receptor has been initially cloned as a death receptor

[2] bound and activated by the apoptotic-inducing mAb

S. Tauzin

Universite Rennes-1, 2 Avenue du Professeur Leon Bernard,

35043 Rennes, France

L. Debure

IRSET, Team ‘‘Death Receptors and Tumor Escape’’,

2 Av du Prof. Leon Bernard, 35043 Rennes, France

J.-F. Moreau

Universite de Bordeaux-2, UMR CNRS 5164,

146 rue Leo Saignat, 33076 Bordeaux, France

P. Legembre (&)

University of Rennes-1, IRSET (Institut de Recherche sur

la Sante l’Environnement et le Travail), Team ‘‘Death Receptors

and Tumor Escape’’, 2 av Prof Leon Bernard,

35043 Rennes cedex, France

e-mail: [email protected]

Cell. Mol. Life Sci. (2012) 69:1261–1277

DOI 10.1007/s00018-011-0866-4 Cellular and Molecular Life Sciences

123

Author's personal copy

designated APO-1 [3]. Consequently, the death-inducing

activity of the antigen APO-1 has been extensively ana-

lyzed for the last 20 years and the pro-apoptotic role of

CD95 is supported by the fact that the cognate ligand of

CD95, the transmembrane CD95L (CD178/FasL) is present

at the surface of activated lymphocytes [4] and natural

killer cells [5] where it orchestrates the elimination of

transformed and infected cells. In addition, CD95L is

found expressed at the surface of neurons [6], corneal

epithelia, and endothelia [7, 8] and Sertoli cells [9] to

prevent the infiltration of immune cells and thus to prohibit

the spread of inflammation in these sensitive organs (i.e.,

brain, eyes, testis, respectively), which are commonly

called ‘‘immune-privileged’’ sites.

Description of physiological ‘‘immune privilege’’ was

later on followed by tumor-mediated ‘‘immune privileged

area’’ since two groups reported that the ectopic expression

of CD95L by malignant cells participated in the elimina-

tion of infiltrating T lymphocytes and thus could play a role

in the establishment of a tumoral site whose access was

denied to immune effector cells [10, 11]. However, these

observations were rapidly challenged since both allogenic

transplant of b-islets [12, 13] and tumor cells [14]

expressing the membrane-bound CD95L led to a much

rapid elimination of the cells as compared to control islets

and malignant cells, respectively, due to an increase in

infiltration of neutrophils and macrophages endowed with

anti-tumoral activities. Confirming these latter findings,

recent studies revealed that in certain pathophysiological

contexts, engagement of the CD95 receptor promoted pro-

inflammatory [15–17] or even pro-oncogenic processes

[18–20]. In the following parts of this review, we will

discuss how CD95 itself and its ligand organize the

implementation of pro- or anti-apoptotic signals.

CD95-mediated apoptotic signaling pathway(s)

Similarly to the TNF-receptor [21], CD95 has been found

expressed at the plasma membrane as a pre-associated

homotrimer [22, 23]. Binding of CD95L or agonistic anti-

CD95 mAbs to CD95 alters both the conformation and the

extent to which the receptor aggregates at the plasma

1 2 3 4 5 6 7 8

1 347 376 542 680 789 851 914 997 1022 1354 2689 nt mRNA

9exons

1 16 174 190 230 317 335 a.a.PROTEIN

PS TM DD

A

B

COOH5

23 4 1

6

Plasma membrane

Cytosol

α

1α

2

α

3

α

4

α

5

α

6

Fig. 1 CD95 structure. a CD95 mRNA consists of 9 exons as

indicated in the upper panel. The CD95 mRNA (upper panel) and

protein (lower panel) are depicted. Numbers on the upper panel stand

for the position of nucleic acids in CD95 mRNA while on the lower

panel, they represent the position of the amino acids in the protein

sequence. The 6 a-helices constituting the CD95-death domain (DD)

are indicated. PS peptide signal, TM transmembrane domain. Num-

bering is applied according to references [2, 59] (NM_000043).

b Nuclear magnetic resonance (NMR) structure of the CD95 death

domain according to 1DDF (Protein data bank) and analyzed using

Jmol version 12.0.18. The rockets stand for the 6 a-helices and the

plasma membrane has been added

1262 S. Tauzin et al.

123


membrane and mounts a signal occurring initially through

protein–protein interactions. In this regard, the intracellular

region of CD95 encompasses an 80-amino-acid-long

stretch designated the death domain (DD) (Fig. 1a, b)

whose structure consists of six amphipathic a-helixes

arranged anti-parallel to one another (Fig. 1b) [24]. Upon

addition of CD95L, CD95 undergoes conformational

modifications of its DD, which elicit the shift of helix 6

and its fusion with helix 5 in order to promote both the

oligomerization of the receptor and the recruitment of the

adaptor protein Fas-associating protein with a death

domain (FADD) [25]. A consequence of the opening of the

globular structure of CD95 is that the receptor becomes

connected through this bridge, which enables an increase in

the magnitude of its homo-aggregation. In addition, this

long helix also allows the stabilization of the complex by

recruiting FADD. Overall, this CD95-DD:FADD-DD

crystal structure not only revealed an attractive mechanism

to explain the formation of large CD95 clusters observed

using imaging or biochemical methods in cells exposed to

CD95L, but also confirmed that alteration of the CD95

conformation was instrumental in the induction of the

signal. However, this elongated C-terminal a-helix favor-

ing the cis-dimerization of CD95-DD [25] was challenged

by Driscoll and Wu’s teams, which did not observe the

fusion of the last two helices at a more neutral pH (pH 6.2)

as compared to the acidic condition (pH 4) used in the

initial study to resolve the CD95-DD:FADD-DD structure

[25]. Consequently, at pH 6.2, association of CD95 with

FADD consisted predominantly of a 5:5 complex that

occurred via a polymerization mechanism involving three

types of asymmetric interactions but without major alter-

ation of the DD globular structure [26, 27]. It is likely that

the low pH condition used in the study performed by Scott

et al. [25] altered CD95 conformation and resulted in the

formation of non-physiological CD95:FADD oligomers.

Nonetheless, we cannot rule out that in vivo, a local

decrease in the intracellular pH may affect the initial steps

of the CD95 signal by promoting the opening of the CD95-

DD, which in turn contributes to the formation of a com-

plex eliciting a sequence of events different from the one

occurring at physiologic pH.

Once docked on CD95-DD, FADD self-associates [28]

and binds and aggregates procaspases 8 and -10, which

are auto-processed and released in the cytosol as active

caspases that cleave many substrates leading to the exe-

cution of the apoptotic program and the death of the cell.

The complex CD95/FADD/Caspase-8/-10 is called DISC

for ‘‘death-inducing signaling complex’’ [29]. Due to the

importance of the DISC formation in the fate of the

cells, it is not surprising that numerous cellular and viral

proteins have been reported to negatively regulate the

formation of this structure, such as FLIP [30, 31] and

PED/PEA-15 [32] that interfere with the recruitment of

caspase-8/-10.

CD95 mutations

Patients exhibiting germinal mutations in APT-1, the gene

encoding the CD95 receptor, develop a syndrome termed

autoimmune lymphoproliferative syndrome type Ia (ALPS,

also called Canale–Smith syndrome) [33–35] (Table 1).

ALPS patients show chronic lymphadenopathy and

splenomegaly, expanded populations of double-negative

a/b T lymphocytes (CD3?CD4-CD8-) and often develop

autoimmunity [33, 34, 36, 37]. In agreement with the

notion that CD95 is a tumor suppressor gene, the ALPS

patients display an increased risk to develop Hodgkin and

non-Hodgkin lymphomas [38]. Predominance of post-ger-

minal center (GC) lymphomas in patients exhibiting either

germ line (Table 1) or somatic CD95 (Table 2) mutations

can be explained by the fact that firstly, inside germinal

centers of the secondary lymphoid follicles, the CD95

signal plays a pivotal role in the deletion of self-reactive

maturating B lymphocytes [39] and secondly, APT-1

belongs to a set of rare genes (i.e., PIM1, c-myc, PAX5,

RhoH/TTF, Bcl-6), which are subjected to somatic hyper-

mutation [40, 41] and thus accumulate mutations that at

one point, may alter their biological function. In addition to

post-GC lymphomas, significant amounts of mutations in

CD95 gene have been found in tumors from various his-

tological origins (summarized in Table 2). An extensive

analysis of the CD95 mutations and their distribution in

APT-1 confirms that with some exceptions, most of them

are gathered in exons 8 and 9 encoding the main part of the

CD95 intracellular region (Fig. 2) [42]. From a biological

standpoint, malignant and ALPS type Ia cells harboring a

heterozygous mutation inside the CD95-DD exhibit resis-

tance towards the CD95-mediated apoptotic signal. Indeed,

in agreement with the notion that CD95 is expressed at the

plasma membrane as a pre-associated oligomer (trimeric

complex) [22, 23], the formation of heterocomplexes

consisting of the association of wild-type with mutated

CD95 will hamper FADD recruitment and thus, will alter

the ignition of the apoptotic signal. Extensive analysis and

positioning of various CD95 mutations described in liter-

ature seem to highlight ‘‘hot-spots’’ of mutation in the

CD95 sequence (Tables 1, 2; Fig. 2a, b). Among these

‘‘hot-spots’’, it is worth noting that together arginine in

position 234, aspartic acid in position 244 and valine

in position 251, account for a significant amount of the

documented CD95 mutations. Indeed, among the 189

mutations annotated in the 335-length CD95 amino acid

sequence, 30 (*16%) are found localized on these three

amino acids (Fig. 2a, b). Strikingly, the pivotal role played

by these amino acids in the stabilization or formation of

CD95-mediated cell signaling in cancer 1263

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Table 1 Germinal mutations in the APT1 gene

Mutations Diseases References Observations

1 Exon 3

Frameshift (-1)

Intron 3 and exon 7:

Splice variants

Exon 9

T225P

Q257X

ALPS Ia [34] Five unrelated ALPS patients

2 Exon 9

D253 (frameshift -2)

L290 (deletion of the last 29 a.a. replaced by YKLHQE)

ALPS Ia [35] Case report of three patients(one sibling and unrelatedpatient)

No surface expression of theL290 mutant

3 Exon 9

K230X (?1 frameshift)

D244Y (father and son)

R234X

ALPS Ia [33] Case report of four patients

4 Exon 1

T12A

Second allele displayed deletion between aa 41 and 96 and exon 6 (no TM)

ALPS Ia in a patient withtype 2 autoimmunehepatitis

[90] Mutation inside exon 1 anddeletion of a part of thePLAD domain

5 Exon 4

R105 W

Exon 9

Y216C

ALPS Ia [91] Autosomal recessive mutations

6 Exon 9

D244 V

ALPS Ia [92]

7 Exon 3

C66R

frameshift

R1X

C47X

Intron 7

S209X

Intron 8

S214X

Exon 9

R234P

D244G

D244Y

T254I

ALPS Ia [93] Cohort of 11 families.

Extracellular mutations (C66R/R1X/C47X) abolished theexpression of the mutatedCD95

8 Exon 3

E63X

Intron 7

splice variant

P201 frameshift

Exon 9

S214 frameshift (?2)

S227 frameshift (?4)

V233L

R234P

G237D

G237S

I243R

T254K

E256K

W265 frameshift

K280 frameshift (-1)

ALPS Ia [37]


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Table 1 continued

Mutations Diseases References Observations

9 Intron 3

P49 splice variant

Exon 3

C57X

D62 frameshift (-1)

Intron 4

T131 splice variant

Intron 6

V174 splice variant

Exon 7

K181(del-11)

Intron 7

P201 splice variant

Exon 9

T225P

T225K

R234Q

R234P

A241D

D244V

Q257X

Q260X

L278X

I294S

ALPS Ia [46] Cohort of 155 families

60 Individuals harbor a CD95mutation.

17 different mutations.

Penetrance of one or moreALPS features was 88% forindividuals with intracellularmutations versus 18% forindividuals withextracellular mutations.

Exon 9 encompasses 59% ofmutations causing ALPS

10 Exon 7

K181 (del-11)

P201 frameshift

Exon 9

T225P

R234X

D244V

T254I

E256G

Hodgkin and non-HodgkinLymphomas associatedwith ALPS type Iapatients

[38, 94] Large-scale family case report.

223 members of 39 families,130 individuals possessedheterozygous germline Fasmutations. ALPS patientsdisplayed an increased riskof non-Hodgkin andHodgkin lymphomas (14and 51 times greater,respectively) as compared togeneral population

11 Exon 9

R234Q

ALPS type Ia/HodgkinLymphomas

[95] Family case report

12 Germline mutations (ALPS type Ia)

K181X

P201X

S214X

Somatic mutations (ALPS Type III)

W173X

P201X (3 cases)

S214X

D244V

ALPS Type Ia/Type III [96] Mutation originated inhematopoietic stem cells:6/6 in double negative Tcells from ALPS Type III

0/5 in healthy subjects

13 Exon 9

I246S

ALPS Ia [97]

14 Exon 3

H95R

Exon 7

E195X

Intron 7 (splice mutation/premature stop)

E202X (5 patients)

Exon 8

N207X (del-8)

L208X (del-2)

Exon 9

D244N

V204 (del-2)

D212X (del-5)

Autoimmunelymphoproliferativesyndrome (ALPS typeIII)

[98] 12/31 of double-negative Tcells displayed CD95mutations


123


Table 2 Somatic mutations in the APT1 gene

Mutations Diseases Mutation frequencies References Observations

1 Exon 9

Y275S

D253Y (2 patients)

K235R

N264H

Multiple myelomas Among 54 patients, 6 did notexpress CD95 and 5–48(10%) displayed mutation.

[99]

2 Exon 2

A(-1)T

Exon 4

C119 (?1) frameshift

Exon 6

L164F

P167L

Exon 7

T182I

L199X

Exon 8

L208X

Exon 9

D244V

N248K (2 patients)

E256K

L262F

K283N

Intron 7

splice variant

Intron 8

splice variant (2 patients)

Non-Hodgkin lymphomas 16/150 non-Hodgkinlymphomas (11%)

3/5 (60%) mucosa-associatedlymphoid tissue (MALT)-type lymphoma

9/43 (21%) diffuse LargeB-cell lymphomas

2/33 (6%) follicle centerlymphomas

1/2 (50%) anaplastic largecell lymphoma

1/17 (6%) B-chroniclymphocytic leukemia

0/5 Burkitt lymphoma

0/9 mantle cell lymphoma

0/35 peripheral T-celllymphoma

[100] Missense mutations withinexon 6 and 7 wereassociated with loss ofheterozygosity (LOH)

3 Exon 9

L213 (frameshift -20)

Adult T cell leukemia Found in an ATL cell line(designated KOB)

[101]

4 Exon 9

A241T

N250S

V251I

Cutaneous malignantmelanoma

3/44 (6.8%) [102] LOH for two patients

5 Exon 6

C162R (2 patients)

Exon 9

V251I (8 patients)

N237K

D244 (?1) stop in 245

Bladder carcinomas 12/43 (28%) [103] 11/12 mutations found inmuscle-invasivetransitional cellcarcinomas

Hotspot mutation in V251

6 Exon 9

I300Va

E323Xa

Hodgkin and Reed–Sternberg lymphomas

1/10 patients (10%) [104]

7 Exon 4

N102S

Exon 6

C162R

Exon 9

N239D

Squamous cell carcinoma 3/21 (14%) burn scar-relatedsquamous cell carcinomadisplayed mutated CD95.

2/6 (33%) with lymph nodemetastasis.

1/15 (7%) without metastasis.

0/50 conventional skinsquamous carcinoma

[105] Mutations are found inmetastatic lesions


123


Table 2 continued


8 Exon 8

Splice variant (deletion codons202–210) 9 6 thyroid lymphoma (TL)and 2 chronic lymphocytic thyroiditis(CLTh)

Exon 9

Y216X

E240V

D244N

D244H

Frameshift exon 9 (?1 codon 285) (9 TLand 1 CLTh)

Thyroid lymphomas 17/26 thyroid lymphomas(65.4%) (consisting of 5/10diffuse large B-celllymphoma; 6/8 low-gradeMALT; 6/8 follicle centercell lymphomas)

3/11 Chronic lymphocyticthyroiditis (27.3%)

[106] Mutation hot-spots in thyroidlymphomas

9 Exon 2

K23R

Exon 3

G50S

Exon 5

D152X

Exon 6

V172A

Exon 9

V229A

284 (Insertion ?1) frameshift

Mucosa associatedlymphoid tissue(MALT)-typelymphomas

4/5 mucosa associatedlymphoid tissue (MALT)-type lymphomas

1/3 DLBLs

[107] Patients display more than 1mutation per gene

10 Exon 9

T254Ab

Q260X

Q260N

N286S

High-grade prostaticintraepithelial neoplasia(HGPIN) and prostaticcancer

4/27 of HGPIN (14.8%) [108]

11 Exon 9

N239D (2 patients)

E240G

D244V

R263H

Gastric cancers 5/43 (11%) [109]

12 Exon 9

D210G

A221G

N239S

H266L

K271M

D276G

I279V

I302V

Testicular germ cell tumor 9/24 patients (37.5%)

A total of 11 mutations werefound in 10 lesions fromthe 9 patients (3 silentmutations)

[110] All mutations in CD95 exon 9were associated with aloss-of-function

13 Exon 2

T24I

Exon 3

C69Y

Exon 4

E98G

Exon 7

Frameshift (-1) E195X

Exon 8

Frameshift (-1) I206X

Exon 9

T203P

Mycosis fungoide (MF)(cutaneous T-celllymphoma)

6/44 (13%) [111] The mutation in CD95 couldexplain the commonresistance of MF tochemotherapy


123


intra and inter-bridges between CD95 and FADD may

explain these ‘‘hot-spots’’ since for instance, both R234 and

D244 contribute to the homotypic aggregation of the

receptor and the FADD recruitment [24]. Nevertheless, the

observation of death domain ‘‘hot-spots’’ is in contradiction

with the study of Scott and colleagues [25] demonstrating

that the region of the CD95-DD interacting with the

FADD-DD spreads on a disperse surface through weak

binding affinities. Most ALPS type Ia patients affected by

malignancies do not undergo a loss of heterozygosity

(LOH), which let us to hypothesize that this heterozygous

configuration may promote carcinogenesis [43, 44]. Sup-

porting this assumption, we demonstrated that although

conservation of a wild-type allele failed to transmit the

apoptotic signal, it was sufficient and mandatory to elicit

non-apoptotic signals such as NF-jB, MAPK [43, 44],

whose inductions promote invasiveness of tumor cells

[42, 45]. Mutations found in the intracellular CD95-DD

exhibit a higher penetrance of the ALPS phenotype fea-

tures in mutation-bearing relatives than extracellular

mutations, which suggest that these latter mutants require

additional dysfunctions to efficiently abrogate the CD95-

mediated apoptotic signal [46]. Alternatively, it could be

tempting to speculate that in contrast to mutations inside

the death domain, other CD95 mutations somehow prevent

the apoptotic signal but fail to promote non-apoptotic ones,

which may contribute to the disease progression.

Plasma membrane and CD95 signal

In addition to down-regulation of CD95 or accumulation of

heterozygous mutations, the plasma membrane distribution

Table 2 continued


14 Exon 4

C119 frameshift (-1)

Exon 6

S154 frameshift (-1)

Exon 9

A285 frameshift (?1) 2 cases

I246V

L287P

Nasal NK/T-cell lymphoma 7/14 (50%) [112] Insertion in polyA tract fromnucleotide 1,088–1,094(A285 frameshift):mutation hot spot

15 Exon 3

G66D

P84S

Exon 9

Q228X

N250S

Non-small cell lung cancer(NSCLC)

4/80 in NSCLC (5%)

3/43 (7%) in NSCLC withoutmetastasis.

1/37 (2%) in NSCLC withmetastasis

[113] All mutations found inNSCLC patients withmetastasis were onlydetected in the metastaticlesion

16 Exon 2

T27I

S3F

S16P

Intron 3

Splice variant

Intron 8

Splice variant

Exon 9

D244 N

Q260X

Mucosa associatedlymphoid tissue(MALT)-typelymphomas

7 mutations found in 5patients

1/18 (5.6%) Mucosaassociated lymphoid tissue(MALT) lymphomas

4/28 (14.3%) diffuse largeB-cell lymphoma(DLBCL)

[114] Mutations in exon 2 do notimpair the transmission ofthe CD95-mediatedapoptotic signal in T-47D(breast) and Jurkat(lymphocyte) cells

17 50UTR Diffuse large B celllymphomas (DLBCL)

3 of 66 [115]

18 RNA editing

Frame shift (?1)

K284 frameshift (?1)

Systemic lupuserythematosus (SLE)

Mutation accounted for 11%of CD95 cDNA clones inSLE patients and thefrequency of mutant cloneswas less than 1% inhealthy subjects

[116] Transcriptional mRNAediting. Not found in otherhuman death receptormRNAs such as DR5, DR6or TNF receptor 1(TNFR1) or in mouseCD95 mRNA

a Amino acid numbers have been modified according to the regular amino acid annotationb Threonine 256 depicted in this study corresponded in fact to amino acid 254


123


A

Y275FInternalization (Lee et al, EMBO J, 2006)

TM

Q257K(Beneteau et al, Cancer Research, 2007)

I297D

Open conformation (Scott et al, Nature, 2009)

Mutation/a.a.

CD95L interactions

1 2 3 4 5 6 7 8 9 1011121314

α 1

α 4

α 2

α 3

α 5

α 6

P201

E202

D244

V251

R234

A285

B

TM

Extracellular

Intracellular

Missense mutation

Nonsense mutation

Frameshift and splice variant.

Missense mutation

Nonsense mutation

Frameshift and splice variant.

Somatic

Germinal

Amino acids involved in the binding of CD95L (Starling et al, 1998)

Humanized Lprcg mutation(V238N)

TM

Q257K(Beneteau et al, Cancer Research, 2007)

Transmembrane region

Nucleotides in blue indicate α-helix constituting the CD95-DD

PS

Y275FInternalization (Lee et al, EMBO J, 2006)

I297D open conformationScott et al, Nature , 2009.

N-glycosylation sites (Shatnyeva et al, 2011)

1 5 10 15 1 5 10 15 20

atg ctg ggc atc tgg acc ctc cta cct ctg gtt ctt acg tct gtt gct aga tta tcg tcc aaa agt gtt aat gcc caa gtg act gac atc aac tcc aag gga ttg gaa ttg agg aag act

M L G I W T L L P L V L T S V A R L S S K S V N A Q V T D I N S K G L E L R K T

25 30 35 40 45 55 60

gtt act aca gtt gag act cag aac ttg gaa ggc ctg cat cat gat ggc caa ttc tgc cat aag ccc tgt cct cca ggt gaa agg aaa gct agg gac tgc aca gtc aat ggg gat gaa cca

V T T V E T Q N L E G L H H D G Q F C H K P C P P G E R K A R D C T V N G D E P

65 70 75 80 85 90 95 100

gac tgc gtg ccc tgc caa gaa ggg aag gag tac aca gac aaa gcc cat ttt tct tcc aaa tgc aga aga tgt aga ttg tgt gat gaa gga cat ggc tta gaa gtg gaa ata aac tgc acc

D C V P C Q E G K E Y T D K A H F S S K C R R C R L C D E G H G L E V E I N C T

105 110 115 120 125 130 135 140

cgg acc cag aat acc aag tgc aga tgt aaa cca aac ttt ttt tgt aac tct act gta tgt gaa cac tgt gac cct tgc acc aaa tgt gaa cat gga atc atc aag gaa tgc aca ctc acc

R T Q N T K C R C K P N F F C N S T V C E H C D P C T K C E H G I I K E C T L T

145 150 155 160 165 170 175 180

agc aac acc aag tgc aaa gag gaa gga tcc aga tct aac ttg ggg tgg ctt tgt ctt ctt ctt ttg cca att cca cta att gtt tgg gtg aag aga aag gaa gta cag aaa aca tgc aga

S N T K C K E E G S R S N L G W L C L L L L P I P L I V W V K R K E V Q K T C R

185 190 195 200 205 210 215 220

aag cac aga aag gaa aac caa ggt tct cat gaa tct cca acc tta aat cct gaa aca gtg gca ata aat tta tct gat gtt gac ttg agt aaa tat atc acc act att gct gga gtc atg

K H R K E N Q G S H E S P T L N P E T V A I N L S D V D L S K Y I T T I A G V M

α-1

225 230 235 240 245 250 255 260

aca cta agt caa gtt aaa ggc ttt gtt cga aag aat ggt gtc aat gaa gcc aaa ata gat gag atc aag aat gac aat gtc caa gac aca gca gaa cag aaa gtt caa ctg ctt cgt aat

T L S Q V K G F V R K N G V N E A K I D E I K N D N V Q D T A E Q K V Q L L R N

α-2 α-3 α-4

265 270 275 280 285 290 295 300

tgg cat caa ctt cat gga aag aaa gaa gcg tat gac aca ttg att aaa gat ctc aaa aaa gcc aat ctt tgt act ctt gca gag aaa att cag act atc atc ctc aag gac att act agt

W H Q L H G K K E A Y D T L I K D L K K A N L C T L A E K I Q T I I L K D I T S

850 860 870 880 890 900 910 920 930 940 950

α-5 α-6

305 310 315

gac tca gaa aat tca aac ttc aga aat gaa atc caa agc ttg gtc tag

D S E N S N F R N E I Q S L V *

970 980 990 1000

Fig. 2 Extensive analysis of the loss-of-function CD95 mutations.

a Mutations reported in Tables 1 and 2 have been placed in the

amino-acid sequence of CD95. The 6 a-helices constituting the death

domain and amino acids implicated either in the CD95/CD95L

interaction or the receptor glycosylation are depicted. b Schematic

distribution of CD95 mutations in the CD95 protein. The number of

mutations per amino acid is depicted on CD95 amino-acid sequence.

The death domain and its 6 a-helices are reported


123


of CD95 may also represent an alternative way for tumor

cells to circumvent the apoptotic signal. Indeed, plasma

membrane is a heterogeneous lipid bilayer comprising

compacted or ‘‘liquid-ordered’’ domains called microdo-

mains, lipid rafts or detergent resistant microdomains

(DRMs), which are described as floating in a more fluid or

liquid-disordered 2-D lipid bilayer. Despite the fact that the

composition and the kinetic of formation and disappear-

ance of these plasma membrane structures remain poorly

understood mostly due to limitation in the methodologies

used to characterize them, the lipid rafts play a crucial role

in the modulation of the initial steps induced by death

receptor signaling. For instance, it has been elegantly

reported that while CD95 is mostly excluded from lipid

rafts in activated T lymphocytes, the TCR-dependent re-

activation of these cells leads to the rapid distribution of

the death receptor into lipid rafts [47]. This CD95 com-

partmentalization is crucial to decrease in the apoptotic

threshold leading to the clonotypic elimination of activated

T-lymphocytes through activation of the CD95-mediated

apoptotic signal [47]. Similarly, the reorganization of

CD95 into DRMs can occur independently of its ligand

upon addition of certain chemotherapeutic drugs (e.g., rit-

uximab [48], resveratrol [49, 50], edelfosine [51–53],

aplidin [54], perifosine [53], cisplatin [55]). The intimate

molecular cascades that underlie this process remain to be

elucidated. Nevertheless, the current evidence lead us

to postulate that alteration of intracellular signaling

pathway(s) (e.g., the PI3K signal [51, 56]) may change

biophysical properties of the plasma membrane such as the

membrane fluidity, which may mimic the CD95L-induced

initial steps in promoting the aggregation and clustering of

CD95 into large lipid raft-enriched platforms, which in turn

favor DISC formation and the induction of the apoptotic

program [57]. On the other hand, these signaling pathways

may exert post-translational modifications of the death

receptor itself and thereby may promote or prevent its

redistribution into lipid rafts.

Regulation of the CD95 plasma membrane distribution

Plasma membrane distribution of CD95

Accumulation of CD95 mutations is not the only way by

which malignant cells may impede the extrinsic signaling

pathway elicited by immune cells or chemo- and radio-

therapeutic regimens. Post-translational modifications in

the intracellular tail of CD95 such as reversible oxidations

or covalent attachment of a palmitic acid have been

reported to alter the plasma membrane distribution of

CD95 and thereby its subsequent signaling pathway

(Fig. 3). For instance, S-glutathionylation of mouse CD95

at the cysteine residue 294 promotes the clustering of

CD95 and its distribution into lipid rafts [58] (Fig. 3). This

amino acid is conserved in the human CD95 sequence and

corresponds to cysteine residue at position 304 (C288 in

Internalization

Lipid Rafts

Palmitoylation

S-gluthationylation

α 1

α 4

α 2

α 3

α 5

α 6

P201

E202

D244

V251

R234

A285

TMIntracellular

C288

C183

Y275

S-nitrosylation

SDS-stable

Microaggregates

AP-2 Binding

DISC

NON-APOPTOTIC

SIGNALS (i.e., NFκκB, MAPK, PI3K)

APOPTOSIS

?

?

?

Extracellular

Fig. 3 Post-translational modifications and CD95 internalization.

The main mutations described in Tables 1 and 2 have been placed in

the amino-acid sequence of CD95. The 6 a-helices constituting the

death domain are depicted. CD95 internalization (AP-2 binding)

hampers the implementation of non-apoptotic signaling pathways

while it elicits apoptosis. Question marks indicate experiments that

remain to be conducted to decipher whether similarly to internaliza-

tion, CD95 palmitoylation, S-nitrosylation and S-glutathionylation

inhibit the CD95-mediated non-apoptotic outcomes. Black arrows

indicate activations; red lines (dashed and continuous) stand for

inhibitions


123


Figs. 2a, 3 whose numbering takes into consideration the

subtraction of the 16-amino acid peptide signal [2, 59]).

Interestingly, Janssen-Heininger Y.M. and colleagues [58]

emphasize that the death receptor glutathionylation occurs

downstream the activation of caspase-8 and -3 whose cat-

alytic activity processes and inhibits the thiol transferases

glutaredoxin 1 (Grx1), an enzyme implicated in the den-

itrosylation of proteins. The consequence of Grx1

inactivation is the accumulation of glutathionylated CD95,

which cluster into lipid rafts sensitizing cells to the CD95-

mediated apoptotic signal. Based on these findings and

counterintuitively, we conclude that caspase-8 activation

occurs prior to aggregation of CD95 and its redistribution

into lipid rafts, which both are mandatory to form the DISC

and subsequently to activate large amounts of caspase-8.

To conciliate these observations, activation of caspase-8

has been reported to occur in a two-step process. First, an

immediate and faint amount of activated caspase-8 (\1%)

is generated when CD95L interacts with CD95 that

orchestrates acid sphingomyelinase (ASM) activation,

ceramide production and CD95 clustering that second,

promote DISC formation and the outburst of caspase-8

processing essential to mount the apoptotic signal [60].

It is noteworthy that S-glutathionylation is not the only

oxidation modulating the CD95 activity. In this regard,

S-nitrosylation has also been reported to cause an augmen-

tation of the plasma membrane expression and the activity

of CD95 [61]. S-nitrosylation of cysteine residues 199

(corresponding to the residue C183 in Figs. 2a, 3) and 304

(C288) in colon and breast tumor cells leads to the redis-

tribution of CD95 into DRMs, the formation of the DISC

and the transmission of the apoptotic signal [61] (Fig. 3).

Two reports have brought to light that covalent coupling

of a 16-carbon fatty acid (palmitic acid) to cysteine residue

at position 199 (C183) elicits the redistribution of CD95

into DRMs, the formation of SDS-stable CD95 microag-

gregates, which resist to denaturing and reducing

treatments and the internalization of the receptor [62, 63]

(Fig. 3). These molecular steps remain to be more finely

ordered but they play critical role in the implementation of

the apoptotic signal (discussed below).

Of note, similarly to S-nitrosylation, both the afore-

mentioned S-glutathionylation at C304 (C288) and

palmitoylation at C199 (C183) promote the partition of

CD95 into lipid rafts and enhance the subsequent apoptotic

signal (Fig. 3). Further investigations addressing if these

post-translational modifications are redundant and occur

simultaneously in dying cells or if they are elicited in a cell

specific and/or in a micro-environmental-specific manner

would be of a great interest to better understand the

molecular mechanisms used by tumor cells to overcome

these post-translational alterations and thereby to resist to

the extrinsic signaling pathway.

Receptor endocytosis

Using an elegant magnetic method to isolate receptor-

containing endocytic vesicles, it has been shown that CD95

is promptly found associated with endosomal and lyso-

somal markers when incubated with an agonistic anti-

CD95 mAb [64]. In addition, expression of a CD95 mutant

in which the tyrosine 291 (Y275 in Figs. 2a, 3) is changed

to phenylalanine does not impinge on FADD binding but

compromises the CD95L-mediated CD95 internalization

occurring through an AP-2/clathrin-driven endocytic

pathway [64]. More strikingly, expression of the internal-

ization defective CD95 mutant (Y291F) abrogates the

transmission of the apoptotic signal while it fails to alter

the non-apoptotic signaling pathways (i.e., NF-jB and Erk)

and even promotes them (Fig. 3). Overall, these findings

provide insight into the presence in the death domain of a

region interacting with AP2 and promoting a clathrin-

dependent endocytic pathway in a FADD-independent

manner. Regarding the role of palmitoylation in the

receptor internalization, the interplay between lipid alter-

ation and AP2/clathrin-driven internalization of CD95

remains to be elucidated.

Implementation of a CD95-mediated non-apoptotic

signaling pathway

CD95L promotes carcinogenesis

Increase rate in malignancies after organ transplantation

represents the major cause of cancer-related mortality in

immunomodulated transplant recipients [65] and provides

the basis for the crucial role played by the immune system

in tumor surveillance. Since among the weapons set at

the disposal of immune cells, CD95L contributes to the

elimination of pre-tumoral cells, it is envisioned that pre-

tumoral cells escaping the immune surveillance will be

shaped to develop resistance to the CD95, a process that

has been termed immunoediting [66]. In other words, the

imprinting of the immune system on pre-tumoral cells

could ultimately select malignant cells with increased

resistance towards the CD95L-induced signal (e.g., down

modulating CD95, expressing dominant negative mutants

of CD95). As heterozygous ‘‘dominant negative’’ muta-

tions of CD95 prevent the CD95-mediated apoptotic signal

triggered by ‘‘weak agonistic’’ molecules such as agonistic

antibodies but do not abrogate the non-apoptotic signals

induced by highly aggregated CD95 ligand (e.g., mem-

brane-embedded CD95L) [67], it is conceivable that these

mutations may increase the apoptotic threshold enough to

switch the signal from apoptotic to non-apoptotic pathways

[44]. Apart from the classical inhibitory effect on apoptosis


123


exerted by the expression of a dominant-negative mutant of

CD95, which may be sufficient to account for carcino-

genesis, we propose that the switch in the CD95 signal (i.e.,

from apoptotic to non-apoptotic signaling pathway) could

account for malignancy progression. In agreement with this

hypothesis, complete loss of CD95 expression [68] and

LOH are rarely observed in malignant cells [42], which

thereby, are still capable to transmit the CD95-mediated

non-apoptotic signals when exposed to CD95L [44]. In this

regard, recent reports confirmed a role of the couple CD95/

CD95L in carcinogenesis through either the activation of

JNK (C-Jun kinase also called stress-activated protein

kinase) [19], NF-jB (nuclear factor-kappa B) [16] or PI3K

(phosphatidylinositol 3-kinase) [20]. Overall, these recent

studies do not rule out the pro-apoptotic function of the

‘‘death receptor’’ CD95 but they emphasize that in certain

pathophysiological contexts, CD95 engagement enables

the transmission of non-apoptotic and even pro-oncogenic

signaling pathways.

From a molecular standpoint, binding of the membrane-

bound CD95L or homemade generated crosslinked soluble

CD95L to CD95 evokes DISC formation building the

platform for the ignition of the apoptotic signal. On the

other hand, we and others recently observed that once

cleaved by metalloprotease, the soluble version of CD95L

(cl-CD95L) fails to induce DISC formation [17, 20] but it

orchestrates the infiltration and accumulation of activated T

lymphocytes in damaged organs of SLE patients through

the formation of a complex that we called the MISC for

motility inducing signaling complex [17]. This complex is

devoid of FADD and caspase-8/-10 but it encompasses the

c-yes src kinase, whose activity contributes to the activa-

tion of the PI3K signaling pathway [17, 20, 69]. These

studies do not only reveal a molecular link between the

‘‘death receptor’’ CD95 and the class I PI3Ks—p110c (also

known as PIK3CG) and d (PIK3CD) isoforms—in T cells

but they also revisit the biological role of CD95L whose

shedding by metalloproteases, frequently over-expressed in

the inflammatory areas, affects the initial events of the

CD95 signaling pathway.

Likewise, decrease in the plasma membrane level of

CD95 or expression of a mutated CD95 allele as observed

in ALPS patients (Table 1) and various malignant cells

(Table 2) impedes the implementation of the apoptotic

signal but does not affect the transmission of non-apoptotic

signals such as NF-jB, MAPK and PI3K [19, 43, 44]

suggesting that these signals stem from different domains

of CD95 or rely on different thresholds to be elicited. One

important question that remains to be addressed is how the

magnitude of the CD95 aggregation controls the formation

of ‘‘death’’- and/or ‘‘motility’’-ISCs. In other words,

although it is well-accepted that the couple CD95/CD95L

can eliminate malignant cells by the implementation of the

DISC or can promote carcinogenesis by fueling inflam-

mation and/or by inducing metastatic dissemination [15–

20, 45], the molecular mechanism(s) underlying the switch

between these different signaling pathways remains enig-

matic. Addressing this question will bring to light new

therapeutical agents able to contain the spreading of

inflammation or to impede carcinogenesis mechanisms at

least in pathologies in which increase in soluble CD95L

amounts has been reported such as cancers (e.g., pancreatic

cancers [70], large granular lymphocytic leukemia and

natural killer cell lymphoma [71]) or autoimmune disorders

(e.g., rheumatoid arthritis and osteoarthritis [72], graft

versus host disease (GVDH) [73, 74] or SLE patients

[17, 75]).

CD95L stoichiometries elicit different cell signaling

pathways

CD95L is a type II transmembrane protein (carboxy-

terminal extracellular region), which belongs to the TNF

(tumor necrosis factor) family. As aforementioned, CD95L

is mainly found at the surface of immune cells where it

contributes to the elimination of infected or transformed

cells through cell-to-cell contact. CD95L is also detected

on macrophages and dendritic cells upon HIV infection

[76] and on surface of epithelial cells in inflammatory

disorders [77]. It is noteworthy that CD95L can be cleaved

by metalloproteases such as MMP3 [78], MMP7 [79],

MMP9 [80] and ADAM-10 (A disintegrin and metallo-

proteinase 10) [81, 82] and shed from the plasma

membrane to be released in the connective tissue and the

bloodstream as a soluble and homotrimeric ligand. Seminal

studies on the metalloprotease-cleaved CD95L have

revealed that this ligand exhibits a homotrimeric stoichi-

ometry [83, 84]. Considering that hexameric CD95L

represents the minimal stoichiometry required to signal

apoptosis [83], the cleaved form of CD95L (cl-CD95L) has

long been considered as an inert ligand competing with its

membrane-bound counterpart (m-CD95L) to antagonize

the transmission of the apoptotic signal [84, 85].

Strikingly, while the soluble form of CD95L generated

by MMP7 (cleavage site inside the 113ELR115 sequence,

Fig. 4) induces apoptosis [79], its counterpart processed

between the serine126 and the leucine127 does not [16, 17,

84]. To explain this discrepancy, one may postulate that the

different quaternary structures of the naturally processed

CD95L underlie the implementation of ‘‘death’’ or ‘‘non-

death’’-inducing signaling complex and their downstream

signals. In agreement with this notion, it has been reported

that soluble CD95L bathed in bronchoalveolar lavages

(BALs) of patients suffering from acute respiratory distress

syndrome (ARDS) undergoes oxidation of two methionine

residues in position 224 and 225 of CD95L (Fig. 4) that


123


enhances the aggregation level of the soluble ligand and

thus its cytotoxic activity [86]. The same authors observed

that the stalk region of CD95L corresponding to amino

acids 103–136 and encompassing the metalloprotease

cleavage sites (Fig. 4) is also instrumental in the multi-

merization of CD95L, which accounts for the damage of

lung epithelium in ARDS patients [86]. Of note, in the

ARDS BALs an additional oxidation occurs at methionine

121 (Fig. 4), which in turn prevents the processing of

CD95L by MMP7 and explains why this cytotoxic ligand

keeps its stalk region [86]. Nonetheless, preservation of this

region in soluble CD95L raises the question if a yet

unidentified MMP7-independent cleavage site exists in

the juxtamembrane region of CD95L, near the plasma

membrane, or if the ligand detected in ARDS patients

corresponds in fact to a full length CD95L embedded in

exosomes [87, 88]. Indeed, this peculiar exosome-bound

CD95L can be expressed by human prostate cancer cells

(i.e., LNCaP) and evokes apoptosis in activated T lym-

phocytes [89].

Although the soluble CD95L cleaved between its ser-

ine126 and leucine127 exhibits no apoptotic activity, this

cytokine fueled inflammation and auto-immunity both in a

lupus-prone mouse model [16] and in systemic lupus ery-

thematosus (SLE) patients [17]. Recent findings on CD95L

emphasize that it may be of a great interest in the future to

finely characterize the quaternary structure of the naturally

processed CD95L found increased in sera of patients

affected by cancers or chronic/acute inflammatory disor-

ders to better comprehend the molecular mechanism

engaged by the ligand and its subsequent biological

functions.

Overall, these results indicate that instead of a mono-

lithic and apoptotic function for the couple CD95/CD95L,

the ratio of transmembrane versus cleaved CD95L or the

plasma membrane amounts of functional CD95 may

account for the induction of apoptotic or non-apoptotic

signals and that way, may favor elimination of transformed

cells or promote carcinogenesis, respectively.

Conclusions

The cellular response to CD95 relies on its expression

level, its alteration by post-translational modifications, the

presence or absence of heterozygous mutations and/or the

stoichiometry of the exposed ligand. However, altogether

these features are not sufficient to foresee the main signal

transmitted by CD95. Fifteen years ago, Stuart and col-

leagues already reported that ‘‘immune privilege’’ does not

only result from the expression of CD95L on epithelial and

endothelial corneal cells but also relies on the site itself in

which the CD95L-expressing cells are transplanted.

Indeed, although transplanted corneas display high rate of

acceptance in the eye, allogeneic corneas grafted hetero-

topically to the skin are rapidly rejected [8]. Likewise, the

group of Nabel shed light on the role of TGF-b in the

CD95-mediated recruitment of neutrophils and thus

strengthened the biological effect of the microenvironment

in the modulation of the CD95 signal [14]. We recently

ascertained that the epithelial-mesenchymal transition

(EMT) causes resistance of tumor cells towards the CD95-

mediated apoptotic signal [68]. Indeed, while tumor cells

displaying an epithelial-like signature were sensitive to the

139

CD95-interacting amino-acids

1 28182 102

TM

QLFHLQKELAELRESTSQMHTASSLEKQIGHPSS-RGS

hCD95L

110

P206 Y218 F275L (gld like)

1

MMP3/7/9 & ADAM10

Self

Assembly Domain

137 - 183

Cleavage sites:

120 130103

STALK REGION

oxidation 2 3

YPQDLVMMEGK218

oxidation

Cis-interaction

Fig. 4 Human CD95L protein structure. Different domains (CD95L

self-assembly/CD95 binding sites/Stalk region) of the type II protein

CD95L are depicted in this scheme. In the stalk region, the amino

acids corresponding to the metalloprotease cleavage sites are depicted

in red. Three targets of oxidation (i.e., methionine) are reported and

their role on the CD95L structure is indicated


123


CD95 signal, the mesenchymal malignant cells resisted to

the transmission of the apoptotic-signaling pathway

through a yet totally unknown process. As TGF-b is the

prototype cytokine contributing to EMT process, it may

correspond to the principal micro-environmental factor,

whose tissue concentration accounts for the CD95 response

both in malignant cells and in innate immune cells. Hith-

erto, it remains to decipher whether the presence of TGF-b

will elicit the expression of factors that hinders the trans-

mission of the CD95 apoptotic signal and promotes the

non-apoptotic signal and/or whether TGF-b will circum-

vent the apoptotic signal and promotes pro-oncogenic

signaling pathways by directly acting on the CD95

expression level and/or the mechanisms of CD95L

cleavage.

Acknowledgments This work was supported by Grants from ANR

(JC07_183182), INCa (projets libres recherche biomedicale), Region

Bretagne, Rennes Metropole and Ligue Contre le Cancer (Comites

d’Ille-et-Vilaine, Comite du Morbihan, Comites de la Dordogne,

Comites des Pyrenees-Atlantiques). S.T. is supported by Ligue Contre

le Cancer (postdoctoral fellowships).

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CD95-mediated cell signaling in cancer: mutations and post-translational modulations